The phosphorothioate drug, WR-2721, which preferentially protects normal tissues from radiation and chemotherapeutic damage, is currently entering clinical trials. Pharmacokinetic data generated in animals following administration of WR-2721 have been obtained using radiolabelled drug. As a prelude to the current submission a new assay for the drug has been developed (see Preliminary Studies). This assay is based on earlier work (1,2,3) and can accurately quantitate and differentiate WR-2721, its dephosphorylated and disulfide forms using fluorescent thiol labelling with monobromobimane and reverse phase HPLC. This assay method allows determination of drug levels at picomole levels in small drug samples (for assessment of purity) and in small tissue samples (1 mg) to allow pharmacokinetic studies in animals and ultimately in humans. We will use the assay method to determine: 1) the intracellular form and site of binding of drug, which may elucidate its mechanisms of action; 2) the rate of its reaction with radicals in protection processes; 3) possible radiation-drug mutagenesis, using in vitro analysis of transformation in 3T3 10T1/2 cells; 4) pharmacokinetics of the drug in normal and tumor tissue of mice and rats; 5) possible alteration of the pharmacokinetics by radiation. This non-invasion method should allow a safe, readily available, and rapid method of analysis which can be used clinically avoiding the problems and hazards of radiolabelled drug use. Ultimately knowledge gained about the site and mechanisms of drug action may lead to development of other, more efficient radioprotective drugs.